The Overexpression Study In Arabidopsis And Kumquat With Fruit Ripening-Specific Gene CsPMEI1 From Citrus

Citrus is the second largest fruit crop in china, and possesses high economic value.The fruit ripening process of citrus is not only related to the formation of fruit quality, but also have a significant impact on the performance of postharvest storage. Molecular biology showed that the fruit ripening was regulated by ripening-related genes. But currently the fruit ripening-related gene of citrus is seldom reported. Therefore, the expression and regulation of fruit ripening-related genes has great significance to explore the molecular mechanisms of fruit ripening and improve fruit quality through biotechnology. In this study, based on the fruit ripening specific gene CsPMEI1 from sweet orange fruit, PMEI1 genes of four kinds of different citrus culivars were isolated. And the evolution relationship and function prediction were analyzed by bioinformatics software. We analyzed the function of citrus fruit specific-gene Cs PMEI1 overexpressed in Arabidopsis by observing the characteristics of biology and botany. The kumquat transgenic plants was successfully obtained by the method of Agrobacterium-mediated, which laid a sound foundation for further analysis of the role of this gene in the formation of.citrus fruit quality. The main results were as follows.(1) The analysis of PMEI1 genes from different citrus species and the phylogenetic analysis of CsPMEI1 gene familyPMEI1 genes of 4 kinds of citrus cultivars including Miyagawa, kumquat, Amakusa and Newhall were isolated. And the nucleotide and amino acid sequences analysis showed that citrus PMEI1 were highly conserved. The family of various species PMEI/InvI genes is divided into three branches by MEGA5.1, and CsPMEI1 belongs to the first branch.(2) The analysis of CsPMEI1 gene overexpressed in ArabidopsisThe previous study obtained CsPMEI1 overexpression transgenic Arabidopsis plants by the combined methods of Agrobacterium-mediated and pollen tube pathway. On this basis, the difference of biological traits of the transgenic Arabidopsis and wild Arabidopsis plants were observed and analyzed by SPSS software. The results showed that the transgenic Arabidopsis was earlier on bolting, flowering and fruiting which were very significantly different between the transgenic Arabidopsis and wild Arabidopsis. And the leaves of transgenic Arabidopsis were a little yellow. The fruit of transgenic Arabidopsis growed in the lower location than the wild Arabidopsis. There is no difference significantly between transgenic Arabidopsis and wild Arabidopsis in terms of plant height. Compared with wild Arabidopsis, the transgenic Arabidopsis had more branches and more fruit which were significant difference with wild Arabidopsis. Besides, the silique length of the transgenic Arabidopsis was shorter and the fruit weight was heavier than the wild Arabidopsis, but the difference was not significant. Therefore, we hypothesized CsPMEI1 was related to early flowering and early fruiting and possibly involved in flowering, fruit development and ripening or other physiological processes.(3) The analysis of CsPMEI gene overexpressed in kumquatThe plant expression vector pBI121-35S-CsPMEI1 was transferred into kumquat genome by the methods of Agrobacterium-mediated. Eventually 181 resistant buds and 13 transgenic plants were obtained. PCR analysis showed that CsPMEI1 gene had been successfully transferred into kumquat genome. The explants sprouting rate was 14.15% and the conversion rate was 1.02%.